Myosin is a major contractile protein present in all nonmuscle eukaryotic cells, but the exact function of myosin in nonmuscle cells is still under study. Recent genetic experiments in Dictyostelium have shown that myosin heavy chains (MHCs) might play a role in cytokinesis, chemotaxis, cell development and specialized cellular functions such as secretion and capping. We have undertaken the cloning of nonmuscle MHC genes and cDNAs to study the function, regulatory mechanism and tissue specific expression of nonmuscle MHCs. The purpose of this study is to clone the cDNA for human nonmuscle MHCs in order to have new tools to examine the role of MHC in cell function. We had already obtained cDNA clones (#302, HL-1, #707) from a human T-lymphocyte lambda gt10 library using different cDNA regional probes from the chicken intestinal epithelial cell MHC. Clone #302 contains a 1.3 kb insert that consists of 1200 nucleotides encoding the first 400 amino acids of a nonmuscle MHC as well as 5' untranslated nucleotides. HL-1 is a 1.9 kb cDNA clone isolated from the same library as #302 and encoding the same portion as that encoded by the 5' portion of pNMHCM2 in macrophages (C.G. Saez et al., Proc. Nad. Acad. Sci. USA 87: 1164-1168, 1989). There was a gap in the sequence of MHC-A between #302 and pNMHCM2. In order to fill in the sequence, we screened a human T-lymphocyte library using three different cDNA probes. Two cDNA clones (ha5- 1, hacal) were isolated, which were 2.4 kb and 1.5 kb in size, respectively, and sequenced partially to complete the gap sequence. The gap sequence of nucleotides and amino acids was very homologous to that of the chicken cell MHC-A (83% identity in nucleotides, 98% identity in amino acids), as compared to human nonmuscle MHC-B (77% in nucleotides, 90% in amino acids).